Digoxin-like fungal glycoside with cytotoxic properties: novel assay and applications

ABSTRACT

The present invention provides methods of isolating cytotoxic metabolites from a fungus, and specific metabolites obtained from such methods. The present invention also provides methods of controlling fungal diseases in plants by treating the plants with cytotoxic metabolites as well as methods of treating cardiac arrhythmia in organisms in need of such treatment by administering the above-noted metabolites to such organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 U.S.C. §119 of U.S.Provisional Patent Application No. 61/129,361, filed Jun. 20, 2008,which application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of bioassays and, moreparticularly, to assays for isolating cytotoxic and/or antimicrobialmetabolites from plant pathogens, and to various uses for such isolatedcompounds.

BACKGROUND OF THE INVENTION

Throughout this application, various patents, published patentapplications and publications, are referenced. Disclosures of thesepatents, published patent applications and publications, in theirentireties, are hereby incorporated by reference into this application.Included among the patents and applications incorporated by referenceare U.S. Provisional Patent Application 60/782,515 and copendingInternational Application No. PCT/SG2007/000071 (which designates theUnited States). In the case of conflict, the present specification,including definitions, will control. Full bibliographic citations forthe publications may be found listed in the List of Referencesimmediately preceding the claims.

Natural products (NPs) are typical secondary metabolites produced byorganisms in response to external stimuli, such as nutritional changes,infection, and adaptive evolution. Several different NPs produced byplants, fungi, bacteria, protozoans, insects and animals have beenisolated as biologically active pharmacophores. Well-known examples ofvaluable NPs used widely in medical and animal health industry includelovastatin (anticholesterolemic agent), cyclosporine A and tacrolimus(immunosuppressive agents), paclitaxel and doxorubicin (antitumoragents), erythromycin (antibiotic), and amphotericin B (fungicidalagent) (Strohl 2000).

A wide variety of actinomycetes have been shown to exhibit significantantifungal activity (Lee and Hwang 2002). Likewise, filamentous fungiare also known to produce a variety of antifungal compounds, includingechinocandins, ergokinin A, sphingofungin, peptaibols, and several othercompounds with a diversity of core structures. A variety of pseudomonadshave been shown to synthesize seed- and crop-protecting antifungals likepyrrolnitrin, syringomycin etc (Rangaswamy et al, 1998). Similarly,extracts of many plants have been shown to contain low-molecular-weightcompounds, which exhibit antifungal activity in vitro. These compoundsinclude a diverse array of secondary metabolites, such as phenolics,saponins, cyanogenic glycosides, cyclic hydroxamic acids,sesquiterpenes, isoflavonoids, sulfur-containing indole derivatives, andmany other compounds (Osbourn, 1999). Flocculosin is a novellow-molecular-weight glycolipid isolated from the yeast-like fungusPseudozyma flocculosa. It is used to control fungal powdery mildewdisease in plants and has also been successfully tested against humanfungal pathogens like C. albicans and Cryptococcus neoformans (Mimee etal, 2005).

In spite of the progress in antifungal therapy, drugs like amphotericinB or triazole have limited use because of their toxicity and/or drugresistance issues (Bagnis and Deray, 2002). Other promising candidatedrugs like Caspofungin have low oral bioavailability (Boucher et al,2004). Hence, there is a need for the isolation or synthesis of newcompounds with different modes of action and low toxicity.

ATP-binding cassette (ABC) transporters, which constitute the largestsuperfamily of proteins known, are able to couple the hydrolysis of ATPto the transport of a variety of substrates either into or out of cells(Ritz et al. 2003). In humans, loss of ABC transporter function has beenimplicated in several pathologies including cystic fibrosis,cholestasis, artherosclerosis, hypoglycemia, hyperbiliruginemia, andmacular dystrophy and degenerative diseases (Pastan and Gottesmann1988). Moreover, the P-glycoprotein class of ABC transporters is able toefflux chemotherapeutic drugs and lipids, resulting in reducedeffectiveness of cancer treatments (Tsuruo et al, 2003). Similarly, ABCtransporters in bacteria are essential for survival and are alsorequired to secrete toxins and antimicrobial agents (Buchaklian andKlug, 2006).

Loss-of-function analysis of ABC3, which encodes a novel multidrugresistance transporter in the cereal pathogen Magnaporthe grisea, showedthat MDR-based efflux plays an essential role in fungal pathogenesis(Sun et al. 2006; PCT International Patent Application No.PCT/SG2007/000071). Abc3-deletion strain of M. grisea has beenclassified as a non-pathogenic mutant. Although it forms the infectionstructures called appressoria, the lack of infectivity in theabc3-delete mutant was correlated to its inability to penetrate the hosttissue, which in turn, was proposed to be due to accumulation of aninhibitory metabolite and/or perturbed redox homeostasis within theappressoria. Further characterization confirmed that Abc3 function isrequired by the blast fungus to withstand cytotoxic and oxidative stressespecially within the appressoria during infection.

SUMMARY OF THE INVENTION

In the present invention, it has been demonstrated that cytotoxicmetabolites can be isolated from a fungus, preferably from theappressoria of the abc3Δ rice-blast fungus Magnaporthe grisea. Inparticular, a cytotoxic metabolite hereinafter referred to as Abc3transporter substrate or “ATS” has been isolated and purified. Moreover,it has been demonstrated herein that ATS shows cytotoxic activityagainst different fungal species. Exogenous addition of ATS preventedthe wild-type Magnaporthe strain from breaching the host surface andshowed enhanced hypersensitive response (HR) in the host plant tissue.Moreover, treatment with the ATS molecule leads to aberrant cytokinesisand morphogenesis in yeasts, such as S. pombe and C. albicans. It alsohas been shown that popular cardiac glycosides like digoxin (includinglanoxin) and ouabain have potential antifungal activity in addition toalready known anti-cardiac arrhythmia activity. Furthermore, it isdemonstrated herein that ATS is functionally related to cardiacglycosides, such as Digoxin, and that exogenous application of ATSreduces heart rate in zebrafish.

Accordingly, the invention provides a method of isolating and guidingthe purification of a cytotoxic metabolite from a fungus, said methodcomprising: preparing an appressorial extract from a fungus; subjectingthe appressorial extract to chromatographic size fractionation to obtainone or more fractions; testing the one or more fractions for cytotoxicactivity; subjecting fractions exhibiting cytotoxic activity to furtherchromatographic fractionation to obtain further fractions; testing thefurther fractions for cytotoxic activity; pooling fractions havingsimilar cytotoxic activity; and subjecting the pooled fractions toliquid chromatography to obtain the isolated cytotoxic metabolite. Theinvention further provides metabolites, particularly ATS, isolated fromthe above methods. The invention also provides methods for controllingfungal diseases in plants, including important crop plants, by treatingsuch plants with the isolated metabolite (ATS) or with cardiacglycosides, such as Digoxin, digoxigenin, and ouabain. Moreover, theinvention provides methods of treating cardiac arrhythmia in anorganism, preferably a vertebrate, by administering the isolatedmetabolite (e.g., ATS) to the organism.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Purification of ATS from abc3Δ appressoria. a) Total crudeappressorial extract from abc3Δ was fractionated by size exclusionchromatography. Arrow and bar indicates fraction number 16 and 17containing highest cytotoxic activity. b) Fraction 16 and 17 fromprevious run were re-fractionated by size exclusion chromatography. Thebar indicates fractions 9 to 16 containing the cytotoxic activity. c)Final step of purification of ATS by liquid chromatography using a C18RP-HPLC column. Arrowhead shows purified ATS. The chromatogram in greenindicates conductivity due to the salt present in the sample.

FIG. 2: ATS is a Digoxin-like glycoside. a) Molecular mass of ATS (m/z780) was identified by mass spectrometric analysis. b) Chemicalstructure of Digoxin (m/z 780) that has a steroid nucleus, sugar sidechain, and a lactone ring. The derivatives from ionized digoxin areindicated with their respective masses. c) ATS (m/z 780) was ionizedfurther by ESI. Daughter ions similar to those from digoxin areencircled.

FIG. 3: Antifungal activity of ATS. a) Cell density of wild-type S.pombe cells was measured in terms of absorbance in presence of wild-typeor abc3Δ appressorial extract. b) ATS treated or untreated wild-type S.pombe cells were stained with calcofluor white (CFW) after 6 h ofincubation. Arrows show septal deposition defect in treated cells.Bar=10 μm. c) S. pombe strain expressing histone-GFP were treated withATS or solvent for 6 h and processed for epifluorescent microscopicdetection. Arrows indicate defects in the nuclear structure. The sameset of cells were also stained with CFW and examined for defects inseptal deposition (arrows). Bar=5 μm.

FIG. 4: Antifungal activity of digoxin, digoxigenin, and ouabain. Celldensity of wild-type S. pombe was measured in terms of absorbance at 600nm in absence or presence of different concentrations of digoxin (a),digoxigenin (b) or ouabain (c). The data for the activity of digoxinrepresent mean±SE of at least two independent experiments.

FIG. 5: ATS is a specific efflux target of Magnaporthe Abc3p. S. pombewild-type and strain expressing M. grisea Abc3 were treated with ATS orresidual solvent and were stained with CFW after 6 h. Bar=5 μm.

FIG. 6: ATS leads to pseudohyphal development in Candida albicans. Panel(A) shows CFW-stained wild-type untreated yeast (upper panel) ortrue-hyphal (lower panel) growth of C. albicans. Panel (B) shows C.albicans treated with ATS for 6 h and stained with CFW. Arrows showseptal deposition defect in the treated cells. Bar=10 μm.

FIG. 7: Effect of ATS on M. grisea. a) Wild-type Magnaporthe Guy11strain was germinated in the presence or absence of ATS for 2-3 h andstained with CFW. Bar=5 μm. b) Panel (A) shows appressorial functionassessed as papillary callose deposits (%; white arrows) after 24 h inuntreated or ATS-treated M. grisea, respectively. Asterisk indicates therare callose deposition in ATS-treated appressoria. Panel B shows DICimages of inoculated rice leaf sheath after 30 h. Arrowheads indicateappressoria that lacked invasive hyphae. Bar=10 μm. Panel (C) shows thequantification of the appressorial function (as in A) in untreated orATS-treated M. grisea on barley leaf explants. Data (presented asMean±SE) was derived from three replicates.

FIG. 8: ATS elicits HR-like response in rice. a) ATS-treated oruntreated barley leaf explant was stained with trypan blue and observedunder bright field. Arrowhead shows visible HR-like cell death. b)ATS-treated or untreated rice leaf explant was stained with CeCl₃ andobserved by electron microscopy. Red or white arrows indicate ceriumperhydroxide granules. Red arrows indicate plasmolysis after ATStreatment for 48 h. CW, cell wall; M, mitochondrion; and V, vacuole.Bar=1 μm.

FIG. 9: Digoxin reduces Magnaporthe infection in barley. Detached barleyleaf pieces were inoculated with 100 or 200 conidia per drop in presenceor absence of 200 μM digoxin. The disease reaction was scored on 6 dpi.The data represent observations from 3 independent experiments.

FIG. 10: Effect of ATS on cardiac activity in Zebra fish. Zebra fishembryos were incubated in the presence of ˜415 nM ATS (in fish water) orresidual solvent and observed under bright field microscope to monitorheart development and function over 3 days post fertilization. Bar chartshowing the heart rates of the larvae treated with ATS or Digoxin orresidual solvent. Heart rate was measured as seconds per 20 beats after26 h of treatment. The data represent mean±SE from three independentexperiments.

DETAILED DESCRIPTION OF THE INVENTION

Loss of Abc3, an MDR efflux pump essential for virulence of therice-blast fungus Magnaporthe grisea, leads to reduced viability andnon-pathogenicity due to the accumulation of cytotoxic metabolite(s) inthe infection structures. In embodiments of the present invention, afission-yeast based novel bioassay has been established to monitor andpurify such toxic metabolite(s) from the appressorial contents of theabc3Δ mutant. ATS is the first metabolite identified in M. grisea withinherent cytotoxic activity and shares some properties of cardiacglycosides of therapeutic importance.

In the present invention, it has been demonstrated that a cytotoxicmetabolite can be isolated from a fungus, preferably from the abc3Δappressoria of the rice-blast fungus Magnaporthe grisea. In particular,a cytotoxic metabolite hereinafter referred to as ABC3 transportersubstrate or “ATS” has been isolated and purified. Moreover, it has beendemonstrated herein that ATS shows cytotoxic activity against differentfungal species. Exogenous addition of ATS prevented the wild-typeMagnaporthe strain from breaching the host plant surface while inducinglocal HR-like response in the host tissue. Moreover, the ATS moleculeachieves aberrant cytokinesis and morphogenesis in yeasts, such as S.pombe and C. albicans. Furthermore it has been shown that ATS isfunctionally related to cardiac glycosides, such as Digoxin, and thatexogenous application of excess ATS specifically perturbs embryonicheart function in zebra fish. In addition, digoxin, digoxigenin, andouabain showed antifungal activity similar to that of ATS; and digoxinreduced fungal infection in plants.

In an aspect, the present invention provides a method of isolating andguiding the purification of a cytotoxic metabolite from a fungus, themethod comprising preparing an appressorial extract from a fungus;subjecting the appressorial extract to chromatographic sizefractionation to obtain one or more fractions; testing the one or morefractions for cytotoxic activity; subjecting fractions exhibitingcytotoxic activity to further chromatographic fractionation to obtainfurther fractions; testing the further fractions for cytotoxic activity;pooling fractions having similar cytotoxic activity; and subjecting thepooled fractions to liquid chromatography to obtain an isolatedcytotoxic metabolite. In preferred embodiments, the fungus is the M.grisea rice blast fungus, more preferably the fungus is an M. griseaabc3Δ strain. In preferred embodiments, the cytotoxic activity tested inthe method is cytotoxic activity against S. pombe, but can, in alternateembodiments include, without limitation, cytotoxicity against buddingyeast Saccharomyces cerevisiae, or C. albicans. Other organisms likeNeurospora crassa, or Ustilago maydis can also be used for the assays.

The appressoria can be obtained through techniques familiar to those ofordinary skill in the art. For example, conidia can be harvested fromfungal cultures and allowed to germinate and form mature appressoriausing known techniques. Chromatography columns, nylon membrane filters,and other suitable equipment and apparatus readily familiar andavailable to those of skill in the art can be utilized as appropriate inthe novel methods described herein.

The invention also provides a cytotoxic metabolite obtained by themethods described herein, including the method described above. Inembodiments, the cytotoxic metabolite is ATS. Tandem MS data suggestthat ATS is a digoxin-like cardiac glycoside. The invention alsoidentifies previously uncharacterized antifungal activity of digoxin,digoxigenin, and ouabain.

The cytotoxic metabolite can possess broad antifungal and/orantimicrobial activity, including, but not limited to toxicity againstyeasts, such as, without limitation, S. pombe, C. albicans, buddingyeast S. cerevisiae, and others, or toxicity against a fungus, such as,without limitation, M. grisea.

In an aspect, the invention also provides a method of controlling afungal disease in a plant, said method comprising treating the plantwith a cytotoxic metabolite as obtained and described as above andelsewhere herein or with digoxin, digoxigenin, or ouabain. The plant canbe an important crop plant, such as rice, barley, or other monocot ordicot species. The fungal disease can be one of a number of fungaldiseases, including, without limitation, rice blast. Various otherfungal diseases on crops can be considered for the treatment, as well,including, for example, powdery mildew in cereals, potato late blight,Fusarium head blight of barley and wheat, leaf rust and loose smut ofwheat, and sheath blight of rice.

In embodiments, the treatment of the plant with a cytotoxic metaboliteof the present invention, such as ATS, or with a steroidal glycoside,such as digoxin, digoxigenin, or ouabain, induces a hypersensitiveresponse in the plant. In alternate embodiments, the treatment causesinhibition of host penetration by the targeted fungal pathogen. Methodsof treating plants to achieve disease control are well known in the art,and can include, for example, spraying.

The present invention also provides a method of treating cardiacarrhythmia in an organism in need of such treatment, by administeringthe cytotoxic metabolite obtained and described as herein, to theorganism. The metabolite is preferably ATS. The organism is preferably amammal, more preferably a human. The administration can be by any modeknown to those of ordinary skill in the art. Preferable modes ofadministration are oral and intravenous.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1982); Sambrook et al.,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989); Sambrook and Russell, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001); Ausubel et al., Current Protocols in Molecular Biology (JohnWiley & Sons, updated through 2005); Glover, DNA Cloning (IRL Press,Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes,(Academic Press, New York, 1992); Guthrie and Fink, Guide to YeastGenetics and Molecular Biology (Academic Press, New York, 1991); Harlowand Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1998); Jakoby and Pastan, 1979; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation(B. D. Haines & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir andC. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition,(Blackwell Scientific Publications, Oxford, 1988); Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebra fish book. Aguide for the laboratory use of zebra fish (Danio rerio), 4th Ed.,(Univ. of Oregon Press, Eugene, Oreg., 2000).

EXAMPLES

In light of the preceding description, one of ordinary skill in the artcan practice the invention to its fullest extent. The present inventionis further described by reference to the following Examples, which areoffered by way of illustration and are not intended to limit theinvention in any manner. Standard techniques well known in the art, orthe techniques described below were utilized.

Example 1

Methodology For the Isolation of ATS From abc3Δ Strain of M. grisea

Conidia were harvested from 8 to 9 day old fungal cultures (abc3Δstrain) and suspended in de-ionized water to get a count ofapproximately 1×10⁶ conidia per ml. Magnaporthe abc3Δ and the S. pombeMBY2838 strain discussed herein (including throughout the variousExamples and other disclosure) can be easily made by one of ordinaryskill in the art using routine experimental protocols (detailed in Sunet al., 2006) and the requisite gene sequences available in the publicdomain [Genbank accession #DQ156556 (ABC3) and SPCC663.03 (PMDI)]. Twohundred microlitres each of such conidial suspension was placed on to aglass coverslip and the conidia were allowed to germinate and formmature appressoria over 24 h under high humidity. At the end ofincubation period, the liquid from the coverslips was collected assupernatant. The appressoria on each coverslip were covered with 100 μlof 0.5 M solution of NaCl and incubated for 5 h in dark under humidconditions. Appressorial content together with the hypertonic solutionwas collected and saved as “appressorial extract”. A cell scraper wasused to collect the appressorial debris attached to the coverslips. Suchtotal appressorial extract was lyophilized and the concentratedappressorial extract was filtered through 0.2 μm nylon membrane filterand size-fractionated and desalted using a Hi-Trap column (GE HealthcareLife Sciences, Sweden) as per the manufacturer's instructions. Elutionwas performed with sterile de-ionized water with the flow rate set at 1ml/min. Eluate was collected as 0.5 ml fractions. The whole set up usedfor this chromatographic elution was that for Fast Performance LiquidChromatography using Akta Purifier 10 (Amersham, GE Healthcare, Sweden).Fraction(s) showing cytotoxic activity against fission yeast wasre-loaded onto the same ‘Hi-Trap’ desalting column for furtherseparation. The fraction(s) with the same cytotoxic activity wascollected, pooled, and loaded onto a C18 reverse phase HPLC column(Phenomenex, USA). The mobile phase used for elution was 30%acetonitrile with 0.1% formic acid and the elution was carried out underisocratic conditions with 0.5 ml/min flow rate and 0.5 ml fractionvolume. The fraction corresponding to a single peak was ascertained topossess the characteristic cytotoxic activity and was subsequently usedas purified ATS for further characterization and molecularidentification.

Example 2

In vitro Analysis of ATS Antifungal Activity Against Fission Yeast

Approximately, 3 μl of 1×10⁷ cells/ml from overnight grown wild-type S.pombe culture MBY104 or MBY2838 strain expressing M. grisea ABC3(pmd1::URA4; MgABC3⁺) was inoculated in 150 μl fresh YES medium in a 96well plate. The cells were incubated at 25° C. on a rocking platform inpresence of 50 μl of de-ionised water (untreated) or 10 ng of purifiedATS (treated). Cell density of untreated or treated wild-type yeastcells was checked in terms of absorbance after every one hour over 5 to6 generations. For microscopic observation of untreated and treatedsamples, the cells were harvested, washed, stained with calcofluor whiteafter 6 h of incubation, and examined by epifluorescent illumination(360 nm excitation) on an Olympus IX71 microscope. Effect of ATS onkaryogamy or mitosis was studied by using an S. pombe strain MBY816;(Wang et al., 2002), where the cells were treated as described above andGFP epifluorescence examined (488 nm excitation) using an Olympus IX71microscope. Experiments were performed in triplicate and confirmed byseveral biological replicates. The S. pombe strain MBY816 can be easilymade by using sequence information in the public domain (Genbankaccession #SPAC1834.04, Accession no. P09988) and the protocols detailedin Wang et al., (2002).

Example 3

Estimation of Minimum Inhibitory Concentration (MIC) of Digoxin,Digoxigenin, and Ouabain For S. pombe

Approximately, 1×10⁷ cells/ml from overnight grown culture of MBY 104was inoculated in 20 ml fresh YES medium in 250 ml flasks. The cellswere incubated at 25° C. on a shaker in absence or presence of differentconcentrations of digoxin. A stock of 1 mM standard glycosides (SigmaAldrich, USA) was prepared by adding 7.8 mg, 3.9 mg, and 7.28 mg ofdigoxin, digoxigenin, and ouabain, respectively, in 10 ml of 50%ethanol. A working stock of 200 μM solution was prepared by diluting 1mM stock with fresh YES medium. Further dilutions were made from thisworking stock by adjusting total volume with YES to 20 ml. Cell densityof untreated or treated wild-type yeast cells was checked in terms ofabsorbance after every one hour over 5 to 6 generations. Experimentswere performed in duplicate each time and confirmed by severalbiological replicates.

Example 4

C. albicans Growth Inhibition Assays

C. albicans strain SC5314 (a kind gift from Y. Wang, Singapore) wasgrown in YPD broth over night at room temperature. Approximately, 3 μlof 1×10⁷ cells/ml culture was inoculated in 150 μl of fresh YPD mediumdispensed in a 96-well plate. The yeast cells were treated in a similarway as S. pombe above. For induction of hyphal growth in Candida strain,10% calf serum was added to the YPD medium and the cells were grown at37° C. for 6 h with or without ATS. For microscopic observation ofuntreated and treated samples (both yeast as well as hyphae), the cellswere harvested, washed, and stained with calcofluor white after 6 h ofincubation.

Example 5

M. grisea Growth Assays

To study the effect of ATS on germinating wild-type M. grisea (Guy11), 1μl of a conidial suspension (ca. 1×10⁶ conidia/ml) was mixed with 20 μlof water or purified ATS and drop-inoculated onto 0.6% agarose andincubated for 2-3 h. Untreated or treated cells were stained withcalcofluor white, washed and observed using epifluorescence microscopymentioned above.

Example 6 Host Leaf Penetration Assays

Approximately 1000 conidia from the wild-type Guy11 strain per spotinoculation (˜20 μl) were used to test penetration of either rice leafsheath or onion epidermis. Twenty microlitre of sterile de-ionised water(control) or purified ATS (˜5 ng) was mixed with 2 μl of conidialsuspension (approximately 1000 conidia) and inoculated onto rice leafsheath or onion epidermis and incubated for 24-30 h under humidconditions. Fungal invasion of the host tissue was quantified bycounting penetration pegs using aniline blue staining of papillarycallose deposits within the host tissue, and by counting appressoriashowing penetration hyphae (DIC imaging). Callose papillae were observedby epifluorescent illumination (360 nm excitation) on an Olympus IX71microscope.

Example 7 Surface Inoculation Assays on Leaf Explants

A 20 μl drop of sterile de-ionised water or purified ATS was inoculatedonto rice or barley leaf blade and incubated for 48 to 72 h. Barley leafblades incubated for 72 h were tested for cell viability by stainingwith Trypan blue. Rice leaf blades inoculated for 48 h were examined forH₂O₂ production by staining with Cerium chloride (CeCl₃) as describedearlier (Tanaka et al. 2006).

Example 8 Barley Leaf Infection Assay

Approximately 100 or 200 conidia from the wild-type Guy11 strain perspot inoculation (˜20 μl) were used to study disease reaction inpresence or absence of digoxin. Twenty microlitre of sterile de-ionisedwater (control) or standard digoxin (200 μM) was mixed with 2 μl ofconidial suspension (approximately 100 or 200 conidia) and inoculatedonto barley leaf blade and incubated for 5-7 days under humidconditions. Disease reaction was scored by visual observation fortypical disease lesions.

Example 9 Enzyme Linked Immunosorbent Assays For Digoxin or ATS

ELISA tests were performed using a standard set of digoxinconcentrations and anti-digoxin monoclonal antibodies (Sigma Aldrich,USA). Purified ATS (50 μl) or standard digoxin (6 ng to 6 μg) was coatedonto ELISA plate. The wells were later blocked overnight at 4° C. with10% calf serum in 1×PBS containing 0.05% Tween 20. Monoclonal antibodies(1:5000) against digoxin used as primary Ab were added to the wells andincubated for 2 h. After incubation, the wells were washed 4 times for15 min each with blocking buffer used above followed by incubation withHRP conjugated anti-mouse secondary antibodies. Wells were washed in asimilar way with 1×PBS containing 0.05% Tween 20 after incubation withsecondary Ab for 1 h. Ready to use TMB substrate (Sigma, Aldrich, USA)was added to the wells to test HRP activity. Assays either withoutantigen (digoxin or ATS) or without primary antisera were run inparallel as negative controls.

Example 10 Recording of Cardiac Activity in Zebra Fish Larvae

Zebra fish (Danio rerio) were raised under standard laboratoryconditions at 28° C. The line used was wild-type TU. A workingconcentration of 415 nM ATS was prepared in fish water. Embryos at 0 to1 hpf were incubated in either ATS (100 ng/300 μl) containing water orthe solvent control (prepared by using any other HPLC fraction collectedduring ATS purification) and observed over 3 dpf. Bright field picturesand videos (streaming with time lapse 40 msec per frame, 150 frames over5.7 sec) were taken using Zeiss Axioplan 2 microscope equipped with aCCD camera. The heart rates (in terms of time taken in seconds tocomplete 20 beats) of control and treated larvae were estimated using adigital chronograph.

Example 11

M. grisea abc3Δ Strain Accumulates Cytotoxic Metabolite ATS

Total extracts from Magnaporthe wild-type or abc3Δ appressoria wasisolated and tested for antifungal activity against S. pombe. Celldensity, in terms of absorbance at 600 nm, of cells in presence orabsence of total appressorial extract was measured after every 1 h over5 to 6 generations (15 to 18 h). The growth kinetics showed inhibitoryeffects of the crude extracts from the abc3Δ appressoria as compared tothat from wild-type. This inhibitory activity was used as a tool toguide the purification of the presumable efflux target of Abc3p.Chromatographic fractionation of the appressorial extracts from abc3Δshowed a range of molecules eluting out based on their respective sizes.These molecules were collected in different fractions using an automatedfraction collector. Molecule(s) of very small size (eluted in fractionnumber 16 and 17) (FIG. 1 a) among all the fractions collected was foundto be the most effective in terms of cytotoxicity toward fission yeastcells. The molecules in fraction 16 and 17 were further separated onHiTrap column and tested for their cytotoxic activity against fissionyeast. Fraction number 9 to 16 therein (FIG. 1 b) showed similarcytotoxic activity against yeast and were pooled and purified using C18RP-HPLC column. Liquid chromatographic separation of fraction 9 to 16 onthe HPLC column showed a single prominent UV (220 nm) peak which waseluted in fraction number 12 (FIG. 1 c). Mass spectrometric analysis bysoft ionization of this molecule in fraction 12 indicated a major m/z780 species (FIG. 2 a). Reference and compound library searchesindicated that Digoxin, a cardiac glycoside from foxglove plant, with asteroid nucleus, sugar side chain, and a lactone ring, has a similar m/z780 (FIG. 2 b) (Qazzaz et al. 1996). Tandem mass spectrometric analysisof m/z 780 species from fraction 12 showed daughter ions of various m/zincluding m/z 647, 516, and 391 (FIG. 2 c). Mass spectrometric analysisof Digoxin shows daughter ions including m/z 650, 520, and 390, whichare successive breakdown products of digitoxose molecules. ELISA testsusing monoclonal anti-digoxin antisera confirmed the immuno-reactivityof ATS towards anti-digoxin antibodies. The concentration of ATS inHPLC-purified samples was estimated to be 0.2 ng/gl. In humans, digoxinis effluxed by a P-glycoprotein. By inference, the inhibitory molecule(ATS) present in fraction 12 was therefore considered to be digoxin-likeglycoside.

Example 12

ATS Affects Cytokinesis in S. pombe

In an in vitro bioassay, S. pombe cells were grown in the absence orpresence of ATS or crude abc3Δ appressorial extract and observed over aperiod of 8 to 10 h. Growth kinetics showed that the cell density(OD_(600 nm)) dropped significantly when treated with ATS or crude abc3Δappressoial extract (FIG. 3 a). The cell density started decreasing at 4h of ATS treatment; whereas the yeast cells treated with theappressorial extract from the wild-type Magnaporthe showed cell densitysimilar to the untreated control. Microscopic observation of theATS-treated and calcofluor-stained cells showed that ATS affectsbiogenesis of cell wall and/or septa in S. pombe. ATS-treated cells wereelongated, enlarged in size and showed aberrant and unipolar deposits ofexcessive septum/cell wall material at the cell tip. One of the ends ofthe affected cells showed surplus staining with CFW indicatingderailment of and excess septal deposition at one cell end unlikeuntreated cells (FIG. 3 b). Similarly, S. pombe cells expressingHistone-GFP were challenged with ATS and the assays showed that ATS alsohad a significant effect on nuclear division in S. pombe (FIG. 3 c).Growth kinetics indicated that incubation for 6 h was enough to observethese profound effects of ATS in fission yeast.

Example 13 Digoxin, Digoxigenin, and Ouabain Show Anti-Fungal Activity

Wild-type S. pombe cells were grown in absence or presence of differentconcentrations of digoxin, digoxigenin, or ouabain and growth kineticswas studied over 6-8 h. While untreated cells showed increase in celldensity over 6 h of incubation, digoxin treated cells showed decrease inabsorbance in a dose dependent manner. The minimum concentration ofdigoxin (FIG. 4 a), digoxigenin (FIG. 4 b), or ouabain (FIG. 4 c)required to completely inhibit growth in S. pombe cells was found to bebetween 100 to 200 μM.

Example 14

ATS is Specifically Effluxed by M. grisea Abc3p

S. pombe strain expressing Magnaporthe ABC3 (MBY 2838) was used to testthe effect of ATS. Importantly, the MBY 2838 cells were not affected bythe presence of ATS in the growth medium. Such ATS-treated MBY 2838cells showed normal cytokinesis with normal cell size and shape like theuntreated control cells of S. pombe (FIG. 5). These findings stronglysuggest that ATS is most likely an efflux target of the Abc3transporterin Magnaporthe.

Example 15

ATS Causes Morphological Changes in C. albicans

Wild-type C. albicans strain SC5314 was grown in the absence (untreated)or presence of ATS in order to study its cytotoxic activity. Both yeastand hyphal form of C. albicans were studied in this assay. The cellswere incubated for 6 h with ATS and stained with Calcofluor White.Microscopic studies of the treated yeast cells showed defects inmorphology in terms of elongation and enlargement of the cells. Thetreated yeast cells also showed excess cell wall/septal depositsespecially at the budding site and over the entire periphery. Similarly,ATS showed strong effects on hyphal cells in Candida resulting inshorter and aberrant hyphal extensions that failed to advance in growth.Moreover, excess cell wall staining by CFW was also seen in case ofATS-treated hyphal cells compared to the control or untreated cells(FIG. 6).

Example 16

ATS Prevents Wild-Type M. grisea From Breaching the Host Surface

Germination of M. grisea in presence of ATS showed that it did notinhibit germination and subsequent development into appressorium oninductive surfaces. However, the germ tubes showed morphological defectsupon the addition of ATS, culminating in relatively shorter, curvedmoieties with excessive septal deposits at the point of germ tubeemergence (FIG. 7 a). Successful penetration of onion epidermis or riceleaf sheath by M. grisea was assessed by observing callose depositionand penetration hyphae within the host tissue. Aniline blue staining forcallose deposition after 24 h post inoculation showed that almost 52% ofthe wild-type untreated appressoria had successfully penetrated the hostcells; whereas only 14% of the ATS-treated wild-type appressoria wereable to induce callose deposition in the host tissue. Microscopicanalysis after 30 h revealed that about 60% of the untreated appressoriadeveloped infection hyphae within the host cells; whereas those wereseen in only 3-4% of the ATS-treated appressoria (FIG. 7 b). Theseresults suggest that ATS has a direct inhibitory action and reducesMagnaporthe invasion into the host plants.

Example 17 ATS Elicits Hypersensitive Response in Rice

Exogenous application of ATS or Digoxin to leaf tissue was examined tostudy its effect on the host plant. Cell viability tests using trypanblue staining of rice or barley leaf tissue showed hypersensitivereaction (HR) like visible cell death after 48 to 72 h in case of theATS inoculated tissue in contrast to the control or uninoculated samples(FIG. 8 a). Elevation in the levels of H₂O₂ was studied in treated anduntreated leaf tissues by TEM analysis after staining them with Ceriumchloride (CeCl₃). Both Digoxin- or ATS-treated leaf tissues showedaccumulation of Cerium perhydroxide precipitate in the cell wall andcell membrane. Moreover, the host cells showed plasmolysis upontreatment with Digoxin or ATS, indicating programmed-like cell deathupon treatment. The control leaf tissue, however, did not show anyplasmolysis or increased accumulation of Cerium perhydroxide precipitate(FIG. 8 b). Thus, ATS or Digoxin showed elicitor-like functions andinduced hypersensitive reaction or disease resistance type of responsein the host plants.

Example 18 Digoxin Reduces Blast Disease Symptoms

Detached barley leaf pieces were inoculated with Magnaporthe conidia (ca100) in the presence or absence of digoxin, and the disease reactionscored for lesions after 6 days. The control leaf pieces without digoxinstarted developing disease symptoms on day 3. However, equivalent numberof conidia in the presence of 200 μM digoxin failed to elicit anydisease symptoms. On day 6 post inoculation, the severity of disease inthe presence of digoxin was significantly reduced compared to controlleaves. Importantly, the leaves inoculated with 100 conidia per dropletdid not show any symptoms in the presence of digoxin even after 6 dpi,whereas the inoculum lacking digoxin showed significant disease lesions(FIG. 9). Digoxin (and, similarly, digoxigenin, ouabain, and ATS),therefore, are potentially useful in controlling fungal diseases inplants.

Example 19 Excess ATS Reduces Heart Rate in Zebra Fish

Zebra fish embryos treated with ˜100 ng of ATS showed no obvious effecton the development of the larvae. However, the most prominent andspecific effect was seen on the cardiac rhythm in the ATS-treatedlarvae. The estimation of heart rates (time in seconds taken for 20beats) in the presence of ATS or Digoxin revealed slower heart ratecompared to normal or residual solvent treated samples until 48 hourspost fertilization (hpf). At 26 heart rates were found to be 12.71,14.18, and 13.79 seconds for control, ATS, and digoxin-treated larvae,respectively (FIG. 10). At 48 hpf, the heart rate was estimated to be9.95 and 11.33 seconds for 20 beats for the control and ATS-treatedlarvae, respectively. At concentrations lower than above i.e. 50, 25, or10 ng, the larvae showed decreasing effect of ATS with normal heartrate. Thus, ATS, at nanomolar concentrations, may have a good potentialtherapeutic use in treating cardiac arrhythmia.

In the present invention, we have successfully isolated and purified anovel cytotoxic metabolite, ATS, from M. grisea making it an attractivemodel for drug discovery and for metabolite and toxicological studies.The cytotoxic effect on fission yeast, Candida spps, and aphytopathogenic fungus showed that ATS has an inherent broad spectrumantifungal activity. The fission-yeast based assay used to guidepurification of this cytotoxic compound in the present work, promises tobe a robust and efficient method for screening and/or evaluating noveldrugs.

The specific inhibition of host penetration by the plant pathogenMagnaporthe, and induction of hypersensitive response in the host plantby ATS or digoxin suggests their potential application in agriculture incontrolling fungal disease(s) of important crop plants. It also has beendemonstrated herein that digoxin and related cardiac glycosides, such asdigoxigenin and ouabain, possess significant antifungal activity andthus, are potentially useful as fungicides. Furthermore, our preliminaryresults on the effects of ATS on cardiac activity in zebra fish pointout its potential therapeutic use in treating arrhythmia.

A combination of size exclusion chromatography and fast performanceliquid chromatography (FPLC) was used to purify the ATS metabolite fromM. grisea. The effect of ATS on cytokinesis in Schizosaccharomycespombe, and host penetration by Magnaporthe, were used as bioassays forthe isolation of ATS from abc3Δ mutant. Purified ATS was analyzed byelectrospray ionization-tandem mass spectrometry (ESI-MS/MS) technique.ATS or digoxin showed inhibitory effects on S. pombe likely due tomitotic defects and faulty septal depositions during cell division. S.pombe expressing M. grisea Abc3p did not show any effects of ATS orDigoxin. Interestingly, ATS treatment led to morphogenetic defects suchas cell elongation and restricted hyphal extension in the opportunisticfungal pathogen Candida albicans. ATS-treated M. grisea showed aberrantseptal deposition in the germ tubes and ATS (or Digoxin) treatmentblocked appressorial function of host penetration. Furthermore, ATS orDigoxin induced hypersensitive reaction in rice leaf tissue likely dueto elevated H₂O₂ in epidermal cells. Exogenous supply of excess ATSresulted in slower than normal heart rates in zebrafish larvae.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

It will be appreciated that the methods, fish, organisms, andcompositions of the instant invention can be incorporated in the form ofa variety of embodiments, only a few of which are disclosed herein.Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

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1. A method of isolating a cytotoxic metabolite from a fungus, saidmethod comprising: a) preparing an appressorial extract from a wild typeor mutant fungus; b) subjecting the appressorial extract tochromatographic size fractionation to obtain one or more fractions; c)testing the one or more fractions for cytotoxic activity; d) subjectingfractions exhibiting cytotoxic activity to further chromatographicfractionation to obtain further fractions; e) testing the furtherfractions for cytotoxic activity; f) pooling fractions having similarcytotoxic activity; and g) subjecting the pooled fractions to liquidchromatography to obtain an isolated cytotoxic metabolite.
 2. The methodof claim 1, wherein the fungus is M. grisea.
 3. The method of claim 1,wherein the fungus is an M. grisea abc3Δ strain.
 4. The method of claim1, wherein the cytotoxic activity is cytotoxicity against S. pombe.
 5. Acytotoxic metabolite obtained by the method of claim
 1. 6. The cytotoxicmetabolite of claim 5, wherein the metabolite is ATS.
 7. The cytotoxicmetabolite of claim 5, wherein the metabolite possesses antifungal orantimicrobial activity.
 8. The cytotoxic metabolite of claim 5, whereinthe metabolite possesses toxicity against a yeast species.
 9. Thecytotoxic metabolite of claim 8, wherein the yeast is selected from thegroup consisting of S. pombe and C. albicans.
 10. The cytotoxicmetabolite of claim 5, wherein the metabolite possesses toxicity againsta fungus.
 11. The cytotoxic metabolite of claim 10, wherein the fungusis M. grisea.
 12. A method of controlling a fungal disease in a plant,said method comprising treatment of the plant with the cytotoxicmetabolite of claim
 5. 13. The method of claim 12, wherein the plant isa crop plant.
 14. The method of claim 13, wherein the crop plant is amonocot or a dicot.
 15. The method of claim 14, wherein the monocot isrice.
 16. The method of claim 12, wherein the fungal disease is riceblast.
 17. The method of claim 12, wherein said treatment induces ahypersensitive response in the plant.
 18. The method of claim 12,wherein said treatment causes inhibition of host penetration by thefungal pathogen.
 19. A method of treating cardiac arrhythmia in anorganism in need thereof, the method comprising administration of thecytotoxic metabolite of claim 5 to the organism.
 20. A method oftreating cardiac arrhythmia in an organism in need thereof, the methodcomprising administration of ATS to the organism.
 21. A method ofcontrolling a fungal disease in a plant, said method comprisingtreatment of the plant with ATS or a cardiac glycoside.
 22. The methodof claim 21, wherein the cardiac glycoside is selected from the groupconsisting of digoxin, digoxigenin, and ouabain.
 23. The method of claim21, wherein the plant is a crop plant.
 24. The method of claim 23,wherein the crop plant is a monocot or a dicot.
 25. The method of claim23, wherein the crop plant is rice.
 26. The method of claim 21, whereinthe fungal disease is rice blast and/or other disease caused byphytopathogenic fungi.
 27. The method of claim 21, wherein saidtreatment induces a hypersensitive response in the plant.
 28. The methodof claim 21, wherein said treatment causes inhibition of hostpenetration by the fungal pathogen.